Regulation of target protein activity through modifier proteins

ABSTRACT

The present invention is based on the discovery that a polypeptide containing the JAB subunit or the JAM domain has peptidase activity, e.g., isopeptidase activity. The present invention provides polypeptides and crystalline polypeptides containing the JAM domain and methods of using such polypeptides to screen for agents capable of affecting the peptidase activity of the polypeptides. The present invention also provides methods of using the JAM domain for rational drug design or identifying agents capable of affecting the peptidase activity of the JAM domain.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e)(1) toU.S. Provisional Application No. 60/261,314, filed on Jan. 12, 2001,U.S. Provisional Application No. 60/322,322, filed on Sep. 14, 2001, andU.S. Provisional Application No. 60/322,030, filed on Sep. 14, 2001, allof which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of peptidaseactivity, more specifically deconjugation, removal, or separation of amodifier protein from a target protein, e.g., de-neddylation orde-ubiquitination.

BACKGROUND OF THE INVENTION

[0003] The function of a protein is regulated via various means in acell. One way to regulate protein function is via conjugation anddeconjugation of a modifier protein to a target protein, e.g.,neddylation and de-neddylation or ubiquitination and de-ubiquitination.

[0004] The major route for protein degradation in the nucleus andcytoplasm of eukaryotic cells is via the ubiquitin/26S proteasomepathway. The 26S proteasome comprises two major subparticles: the 20Sproteasome and the 19S regulatory particle. The 20S proteasome is acylindrical structure with an internal cavity that contains thepeptidase active sites. Substrates of the proteasome are inserted intothe cylinder, where they are susceptible to digestion by the peptidaseactive sites of the 20S proteasome. Entry into the 20S proteasomecylinder is governed by the 19S regulatory particle, which caps the endsof the 20S cylinder.

[0005] The 19S regulatory particle binds ubiquitinated substrates andtranslocates them into the inner cavity of the 20S cylinder, where theyare degraded. The 19S regulatory particle can be further subdivided intotwo multiprotein complexes: the base and the lid. The base comprises aset of six ATPases that are thought to unfold substrates and translocatethem into the 20S proteasome. The lid is comprised of a set of eightproteins of unknown function. Biochemical data indicate that thepresence of the lid renders the proteasome selective for degradingubiquitinated proteins, but the basis for this selectivity is not known.

[0006] The lid subcomplex of the 26S proteasome is evolutionarilyrelated to the COP9-signalosome complex, but the significance of thissimilarity has remained unknown. There are reports in the literaturethat 26S proteasome preparations contain a variety of associatedubiquitin isopeptidase activities (Eytan, E., et al., J.Biol Chem.268:4668-74 (1993); Verma, et al., Mol Biol Cell 11:3425 (2000)).However, none of these reports demonstrate that an ubiquitinatedsubstrate can be completely deubiquitinated by purified 26S proteasometo yield unmodified substrate. The failure to detect such a reactionproduct may be due to a tight coupling between the deubiquitination of asubstrate and its subsequent degradation within the internal cavity ofthe 20S proteasome.

[0007] Proteins that are destined for degradation by the ubiquitin/26Spathway are marked by the attachment of a multiubiquitin chain to theside chains of lysine residues on the target protein. The ubiquitinatedprotein is then recognized by the 26S proteasome by a mechanism thatremains poorly understood. Subsequently the ubiquitinated protein isdisengaged from any tightly bound partners, unfolded, and translocatedinto the central cavity of the 20S complex, where it is exhaustivelydegraded by the proteolytic active sites that are present in this innercavity.

[0008] Despite many years of intensive study of this system, it remainsunclear what happens to the substrate-bound multiubiquitin chains thattarget the substrate for degradation. It appears that ubiquitin is notdegraded by the proteasome and in fact is recycled. However it remainsunclear if the ubiquitin chains enter the inner cavity of the proteasomeand emerge unscathed, or are cleaved from the substrate protein prior toor during its translocation into the inner cavity of the 20S. In priorwork (Eytan, E., et al., J. Biol Chem. 268:4668-74 (1993)), it wasdemonstrated that there is an isopeptidase activity or activitiesassociated with the intact 26S proteasome that is able to release freeubiquitin monomers from ubiquitinated substrates that are degraded bythe 26S proteasome. It was demonstrated that this activity is sensitiveto the metal ion chelator 1,10-phenanthroline, but the identity of thepolypeptides that harbors this activity was not established, nor was itestablished that this activity is intrinsic to the 26S proteasome asopposed to being intrinsic to a protein that binds transiently to the26S proteasome. This prior work also fails to disclose that theubiquitin isopeptidase activity is critical to the protein-degradingfunction of the 26S proteasome.

[0009] Nedd8 is an ubiquitin-like protein. Like ubiquitin, it iscovalently linked via its carboxy terminus to the side-chain amino groupof lysine residues in target proteins (referred to as neddylation). Theattachment of Nedd8 to target proteins requires the combined action ofNedd8-activating enzyme composed of Ula1 and Uba3 subunits (analogous toubiquitin-activating enzyme, E1), and Ubc12, which is homologous to theubiquitin-conjugating enzymes (E2s). There is no known requirement foran activity equivalent to the ubiquitin ligase (E3) component ofubiquitination pathways. The Nedd8 modification, like ubiquitination, isprobably dynamic. However little is known about the nature of theenzymes that would cleave Nedd8 from its targets (i.e. deneddylate, alsoreferred to as ‘deneddylation’ or ‘deneddylating’ activity).

[0010] A ubiquitin isopeptidase (USP21) has been shown to be able todeneddylate Cul1-Nedd8 conjugates, and a second enzyme UCH-L3, has beenshown to be able to cleave Nedd8-containing fusion proteins at theC-terminus of Nedd8 to release mature Nedd8 (Nedd8, like ubiquitin, ismade as a precursor with additional C-terminal residues that must beremoved before it can be conjugated to proteins). Despite the fact thatboth USP21 and UCH-L3 can metabolize Nedd8-based substrates, they alsowork on ubiquitin-based substrates, and it remains unclear whether theirbiochemical activity towards Nedd8 is relevant in the context of a cell.

[0011] In contrast to ubiquitin, the attachment of Nedd8 to proteinsdoes not mark them for degradation. Rather, it appears as if neddylationacts to modify protein function, much like phosphorylation. The onlyproteins that have been found to be conjugated with Nedd8 to date arethe cullins. The cullins are a family of six related proteins that binda RING-H2 domain protein to form the catalytic core of multisubunitubiquitin ligases. All cullins examined have been shown to be modifiedby attachment of Nedd8, and neddylation of Cul1 has been shown topotentiate the ubiquitin ligase of the SCF complex within which Cul1resides. Thus, for Cul1-based ubiquitin ligases, neddylation serves as apositive regulator of activity. However, for other cullin-basedubiquitin ligases, the impact of neddylation remains uncertain.

[0012] The COP9/signalosome (hereafter referred to as CSN) wasoriginally identified as a regulator of photomorphogenetic developmentin plants. In seedlings grown in the dark, CSN enables a putativeubiquitin ligase known as COP1 to mediate rapid turnover of thetranscriptional regulatory protein HY5 in the nucleus. In seedlings thathave been exposed to light or in CSN mutants, COP1 redistributes fromthe nucleus to the cytoplasm, thereby stabilizing HY5 and allowing it toaccumulate in the nucleus. HY5, in turn, activates the transcription ofa broad palette of genes that mediate photomorphogenetic development.Although the general physiological role of CSN in photomorphogenesis hasbeen defined, little is known about other potential physiologicalfunctions for CSN, and the biochemical function of CSN remainscompletely elusive. It has been suggested that CSN might play a role inubiquitin-dependent proteolysis, based on the observation that the eightsubunits of CSN share homology to subunits of the lid subcomplex of the19S regulator of the 26S proteasome. However, a similar pattern ofhomology is shared with subunits of the eukaryotic initiation factor-3(eIF3) complex. Besides plants, CSN complexes have been discovered inhuman cells, Drosophila, and the fission yeast Schizosaccharomycespombe. Surprisingly, the budding yeast Saccharomyces cerevisiae does notcontain an apparent CSN complex, but does contain a gene homologous tothe CSN5/JAB 1 subunit of CSN complex, thereby implicating CSN5 as beingthe critical component of CSN.

[0013] There is a need in the art to identify the active domain and siteof peptidase activity, e.g., isopeptidase activity of a protein involvedin deconjugation of a modifier protein from a target protein, e.g.,de-neddylation or de-ubiquitination and use such active site for drugdesign. There is also a need in the art to provide screening methods foragents capable of affecting the peptidase activity, e.g., isopeptidaseactivity of a protein and use such agents to treat relevant conditions.

SUMMARY OF THE INVENTION

[0014] The present invention is based on the discovery that the JABsubunit, more specifically the JAM domain is responsible for thepeptidase activity of a protein, e.g., the protein's ability of cleavinga peptide bond between the carboxy terminus of a modifier protein and afree amino group of a target protein. The present invention providespolypeptides and crystalline polypeptides containing the JAM domain andmethods of using such domain to treat conditions associated withpeptidase activity, especially isopeptidase activity and methods toscreen for agents that are capable of modulating its peptidase activity.The present invention also provides methods of using such domain toidentify or design compounds that are candidates for modulators of thepeptidase activity.

[0015] In one embodiment, the present invention provides a method ofdeconjugating a modifier protein from a target protein. The modifierprotein is conjugated to the target protein via a peptide bond betweenthe carboxy terminus of the modifier protein and a free amino group ofthe target protein. The method comprises contacting the target proteinto a polypeptide comprising a subunit characterized as JAB subunit.

[0016] In another embodiment, the present invention provides a methodfor screening for an agent that affects deconjugation of a modifierprotein from a target protein. The modifier protein is conjugated to thetarget protein via a peptide bond between the carboxy terminus of themodifier protein and a free amino group of the target protein. Themethod comprises incubating in the presence and absence of a test agent,the target protein and a polypeptide comprising a subunit characterizedas JAB subunit, determining the effect of the test agent, wherein anincrease or decrease in the amount of the target protein not conjugatedto the modifier protein caused by the test agent is indicative of anagent affecting deconjugation of the modifier protein from the targetprotein.

[0017] In yet another embodiment, the present invention provides anagent identified by the screening method of the present invention.

[0018] In still another embodiment, the present invention provides amethod of increasing conjugation of a modifier protein to a targetprotein. The modifier protein is conjugated to the target protein via apeptide bond between the carboxy terminus of the modifier protein and afree amino group of the target protein in a cell. The method comprisesinhibiting a polypeptide comprising a subunit characterized as JABsubunit in the cell.

[0019] In another embodiment, the present invention provides a method oftreating a condition selected from the group consisting of neoplasticgrowth, angiogenesis, infection, chronic inflammation, asthma, ischemiaand reperfusion, multiple sclerosis, rheumatoid arthritis, andpsoriasis. The method comprises administering an agent identified by thescreening method of the present invention to a subject in need of suchtreatment.

SUMMARY OF THE FIGURES

[0020]FIG. 1 shows the sequence alignment of human AMSH proteins withhuman JAB1 and Rpn11. Conserved active site residues are boxed.

[0021]FIG. 2 shows the sequence alignment of AMSH, AMSH1, and AMSH2. Thecritical conserved active site residues are boxed.

[0022]FIG. 3 shows that sequence alignment of COP9 subunit 5 9CSN5/JAB1)orthologs from different eukaryotic species reveals conserved histidine,serine, and aspartate residues that are also found in a set ofprokaryotic and archaebacterial proteins.

[0023]FIG. 4 shows the alignment of Rpn11 orthologs (Pad1) and CSN5/JAB1orthologs from different species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates in general to active sitesresponsible for the peptidase activity, e.g., isopeptidase activity of aprotein. It is a discovery of the present invention that a polypeptidecontaining the JAB subunit, more specifically the JAM domain haspeptidase activity, e.g., cleavage of a peptide bond between the carboxyterminus of a modifier protein and a free amino group of a targetprotein. Polypeptides containing the JAB subunit, more specifically theJAM domain can be used to treat conditions associated with the peptidaseactivity, screen for agents capable of modulating the peptidaseactivity, and identify or design compounds that are candidates formodulators of the peptidase activity.

[0025] According to the present invention, a modifier protein can bedeconjugated, removed, or separated from a target protein by exposing orcontacting the target protein to a polypeptide containing a subunitcharacterized as JAB subunit or a domain characterized as JAM domain.The modifier protein is usually associated with a target protein via apeptide bond between the carboxy terminus of the modifier protein and afree amino group of the target protein. A free amino group generallyincludes, without limitation, an amino group of the amino terminus of apolypeptide or the epsilon amino group of lysine residues of apolypeptide.

[0026] According to the present invention, the JAB subunit may be theJAB1 subunit of COP9/signalsome (CSN) as disclosed in FIG. 1 (SEQ ID NO.3) or the Rpn11 subunit of 26S proteasome as disclosed in FIG. 1 (SEQ IDNO. 4). A polypeptide subunit can be characterized as JAB subunit if ithas the sequence characteristics of the JAB1 or Rpn11 subunit. Normallya polypeptide subunit is deemed to have the sequence characteristics ofthe JAB1 or Rpn11 subunit if it contains the JAM domain or is a homologor ortholog of JAB1 or Rpn11 subunit. For example, FIG. 3 shows variousorthologs of JAB1 from different species while FIG. 4 shows variousorthologs of Rpn11 and JAB1 including an alignment of orthologs of Rpn11and JAB1 from different species.

[0027] Polypeptides containing a subunit characterized as JAB subunitcan be any known or to be discovered polypeptides, proteins, orcomplexes thereof. For example, the polypeptides may be a polypeptidecomplex of CSN containing JAB1 subunit, 26S proteasome containing Rpn11,AMSH, AMSH1, AMSH2, or C6-1A. CSN has previously been identified as aregulator of photomorphogenetic development in plants. 26S proteasome isinvolved in various activities, e.g., ubiquitin/26S proteasome pathwaywhile AMSH, AMSH1, and AMSH2 are involved in cytokine signaling, TGF-βsignaling, and survival of hippocampal neurons. C6-1A has been observedto be fused to the T cell receptor alpha chain gene by a chromosomaltranslocation in a patient with T-cell leukemia.

[0028] According to one embodiment of the invention, the deconjugation,removal, or separation of a modifier protein from a target protein isachieved by exposing or contacting the target protein to a polypeptidecontaining a domain characterized as the JAM domain. The JAM domain ofthe present invention includes any domain containing the amino acidsequence of HXHXXXXXXXXXD (SEQ ID NO. 1), with H being histidine, Dbeing aspartate, and X being any amino acid. In one embodiment, the JAMdomain is any domain having an amino acid sequence ofGW(Y/I)H(S/T)HPXXXXXXSXXD (SEQ ID NO. 2), with G being glycine, W beingtryptophan, Y being tyrosine, I being isoleucine, H being histidine, Pbeing proline, S being serine, T being threonine, D being aspartate, Xbeing any amino acid, Y/I being either Y or I, and S/T being either S orT.

[0029] In another embodiment, the JAM domain is from a human protein andhas the amino acid sequence of SEQ ID NO. 2. In yet another embodiment,the JAM domain includes any domain having the same peptidase activity,e.g., isopeptidase activity of a domain containing the amino acidsequence of SEQ ID NO. 1 or SEQ ID NO. 2. In still another embodiment,the JAM domain contains a metalloenzyme, e.g., metalloprotease activesite and provides peptidase activity, e.g., isopeptidase activity.

[0030] One feature of the present invention provides isolatedpolypeptides containing a domain characterized as the JAM domain. Forexample, the present invention provides an isolated polypeptidecontaining the JAM domain which is not surrounded by or adjacent to anyamino acid sequences that are naturally adjacent to such JAM domain.

[0031] Another feature of the present invention provides isolatedcrystalline polypeptides containing the JAM domain. The crystallinepolypeptides can be made by any suitable means known to one skilled inthe art. For example, polypeptides containing the JAM domain can becrystallized by equilibrating saturated solutions of the polypeptideswith salts, volatile organic compounds, and other organic compounds atvarious controlled temperatures.

[0032] Yet another feature of the present invention provides isolatedmonoclonal antibodies that specifically bind to an epitope within theJAM domain. Such monoclonal antibodies can be prepared by any meansknown to one skilled in the art. For example, whole or partial aminoacid sequences of the JAM domain can be used as an antigen to obtainmonoclonal antibodies. In one embodiment, the amino acid sequence usedas antigen contains the histidine or aspartate in SEQ ID NO. 1 or SEQ IDNO. 2.

[0033] Polypeptides containing the JAM domain include all known or to bediscovered polypeptides, proteins, or complexes thereof. For example,Table 1 shows various polypeptides, proteins, or fragments thereof fromdifferent species that contain the JAM domain. TABLE 1JAM-Domain-Containing Proteins From Model Organisms* Bacteria Mostbacteria encode one and some 2-4 paralogs of the DNA repair proteinRadC, which is a distinct version of the JAM domain. In addition:Aquifex aeolicus [aquificales] taxid 63363 gi|15606783|ref|NP_214163.1|(NC_000918) hypothetical prot . . . Mycobacterium tuberculosis H37Rv[high GC Gram+] taxid 83332 gi|15608474|ref|NP_215850.1| (NC_000962)hypothetical prot . . . Synechocystis sp. PCC 6803 [cyanobacteria] taxid1148 gi|16330214|ref|NP_440942.1| (NC_000911) unknown protein [ . . .Deinococcus radiodurans [eubacteria] taxid 1299gi|15805429|ref|NP_294125.1| (NC_001263) conserved hypothe . . .Pseudomonas aeruginosa [g-proteobacteria] taxid 287gi|15595836|ref|NP_249330.1| (NC_002516) conserved hypothe . . .gi|15597298|ref|NP_250792.1| (NC_002516) hypothetical prot . . .Coxiella burnetii [g-proteobacteria] taxid 777gi|10956045|ref|NP_052867.1| (NC_002131) hypothetical prot . . .gi|1070034|emb|CAA63684.1| (X93204) orf 112 [Coxiella burn . . .gi|10956011|ref|NP_052361.1| (NC_002118) orf 169; similari . . . ArchaeaPyrococcus abyssi [euryarchaeotes] taxid 29292gi|14520886|ref|NP_126361.1| (NC_000868) hypothetical prot . . .gi|14521781|ref|NP_127257.1| (NC_000868) hypothetical prot . . .Methanothermobacter thermautotrophicus [euryarchaeotes] taxid 145262gi|15678989|ref|NP_276106.1| (NC_000916) unknown [Methanot . . .Halobacterium sp. NRC-1 [euryarchaeotes] taxid 64091gi|16554503|ref|NP_444227.1| (NC_002607) Uncharacterized c . . .gi|15789943|ref|NP_279767.1| (NC_002607) Vng0778c [Halobac . . .Archaeoglobus fulgidus [euryarchaeotes] taxid 2234gi|11499780|ref|NP_071023.1| (NC_000917) conserved hypothe . . .Aeropyrum pernix [crenarchaeotes] taxid 56636gi|14600889|ref|NP_147414.1| (NC_000854) hypothetical prot . . .Sulfolobus solfataricus [crenarchaeotes] taxid 2287gi|15897071|ref|NP_341676.1| (NC_002754) Hypothetical prot . . .Eukaryotes Saccharomyces cerevisiae (baker's yeast) [fungi] taxid 4932gi|14318526|ref|NP_116659.1| (NC_001138) Suppressor of mut . . . Rpnllpgi|6319985|ref|NP_010065.1| (NC_001136) Hypothetical ORF; . . . RrilpSchizosaccharomyces pombe (fission yeast) [fungi] taxid 4896gi|3334476|sp|P41878|PAD1_SCHPO PROTEIN PAD1/SKS1 >gi| 7493 . . .gi|11281515|pir||T44427 hypothetical protein-fission yea . . .gi|7492119|pir||T37756 jun activation domain binding prote . . .gi|9588467|emb|CAC00558.1| (AL390814) similarity to human . . .Arabidopsis thaliana (thale cress) [eudicots] taxid 3702gi|15224003|ref|NP_177279.1| (NC_003070) c-Jun coactivator . . .gi|15219970|ref|NP_173705.1| (NC_003070) putative JUN kina . . .gi|15237785|ref|NP_197745.1| (NC_003076) 26S proteasome, n . . .gi|15229710|ref|NP_187736.1| (NC_003074) 26S proteasome re . . .gi|15239230|ref|NP_196197.1| (NC_003076) 26S proteasome re . . .gi|15218589|ref|NP_172530.1| (NC_003070) hypothetical prot . . .gi|15221964|ref|NP_175311.1| (NC_003070) hypothetical prot . . .gi|15231308|ref|NP_187338.1| (NC_003074) unknown protein [ . . .gi|5902365|gb|AAD55467.1|AC009322_7 (AC009322) Putative sp . . .gi|5091556|gb|AAD39585.1|AC007067_25 (AC007067) T10O24.25 . . .gi|6573732|gb|AAF17652.1|AC009398_1 (AC009398) F20B24.2 [A . . .gi|15220090|ref|NP_178138.1| (NC_003070) hypothetical prot . . .gi|5902374|gb|AAD55476.1|AC009322_16 (AC009322) Hypothetic . . .Drosophila melanogaster (fruit fly) [flies] taxid 7227gi|17137694|ref|NP_477442.1| (NM_058094) CSN5-P1; Drosophi . . .gi|4732109|gb|AAD28608.1|AF129083_1 (AF129083) COP9 signal . . .gi|6434964|gb|AAF08394.1|AF145313_1 (AF145313) 26S proteas . . .gi|7291779|gb|AAF47199.1| (AE003464) Mov34 gene product [D . . .gi|7301945|gb|AAF57051.1| (AE003774) CG2224 gene product [ . . .gi|7303518|gb|AAF58573.1| (AE003823) CG8877 gene product [ . . .gi|7297828|gb|AAF53077.1| (AE003631) CG4751 gene product [ . . .gi|6752672|gb|AAF27818.1|AF195189_1 (AF195189) yippee inte . . .Caenorhabditis elegans [nematodes] taxid 6239gi|17538322|ref|NP_500841.1| (NM_068440) B0547.1.p [Caenor . . .gi|17553290|ref|NP_498470.1| (NM_066069) F37A4.5.p [Caenor . . .gi|17535703|ref|NP_494712.1| (NM_062311) K07D4.3.p [Caenor . . .gi|17508685|ref|NP_491319.1| (NM_058918) R12E2.3.p [Caenor . . . Homosapiens (human) [mammals] taxid 9606 gi|12734403|ref|XP_011713.1|(XM_011713) COP9 (constitutiv . . . gi|5031981|ref|NP_005796.1|(NM_005805) 26S proteasome-ass . . . gi|7243127|dbj|BAA92611.1|(AB037794) KIAA1373 protein [Ho . . . gi|5453544|ref|NP_006454.1|(NM_006463) associated molecul . . . gi|16158201|ref|XP_055481.1|(XM_055481) KIAA1915 protein . . . gi|1168719|sp|P46736|C61A_HUMAN C6.1APROTEIN >gi| 2135176| . . . gi|4581082|gb|AAD24592.1|AC007292_2(AC007292) R31167_1, p . . . gi|7717235|gb|AAB30469.2| (S72931) T-cellreceptor alpha c . . . gi|14249610|ref|NP_116257.1| (NM_032868)hypothetical prot . . .

[0034] According to the present invention, a polypeptide containing theJAB subunit or JAM domain can additionally include any other amino acidsequences. In one embodiment, a polypeptide containing the JAB subunitor JAM domain has other amino acid sequences that do not interfere orinhibit the peptidase activity of the JAB subunit or JAM domain. Inanother embodiment, a polypeptide containing the JAB subunit or JAMdomain has other amino acid sequences that enhance or facilitate thepeptidase activity of the JAB subunit or JAM domain.

[0035] In yet another embodiment, a polypeptide containing the JABsubunit or JAM domain has other amino acid sequences that are associatedwith or determine the specificity of the peptidase activity of the JABsubunit or JAM domain. In still another embodiment, a polypeptidecontaining the JAB subunit or JAM domain has other amino acid sequencesthat inhibit or decrease the activity of the JAB subunit or JAM domain,and such inhibition can be released by a signal, e.g., second messenger,covalent modification, calcium or phosphorylation.

[0036] The modifier protein of the present invention can be any proteinthat modifies the activity or function of a target protein. In oneembodiment, the modifier protein modifies a target protein throughconjugation and deconjugation to the target protein, e.g., formation andcleavage of a peptide bond between the carboxy terminus of the modifierprotein and a free amino group of the target protein. For example, APG12and URM1 modifies a target protein via forming an isopeptide bondbetween the carboxy terminus of APG12 or URM1 and a free amino group ofthe target protein. A free amino group of a target protein usuallyincludes, without limitation, an amino group of the amino terminus orepsilon amino group of lysine residues of the target protein.

[0037] One major class of modifier proteins is ubiquitin. Proteinsdestined for degradation may be marked by the attachment of amultiubiquitin chain to the side chains of lysine residues of theprotein. Another class of modifier proteins include ubiquitin-likeproteins, e.g., NEDD8, UBL1/SUMO, SMT3H2, SMT3H1, FAT10, Fau,UCRP/ISG15, or UBL5. In one embodiment, the modifier protein of thepresent invention includes any protein containing two glycine aminoacids at its carboxy terminus after being processed.

[0038] The target protein of the present invention can be any proteinwhose activity or function is modified by a modifier protein. In oneembodiment, a target protein is a protein which forms a peptide bondwith a modifier protein, e.g., a peptide bond between the carboxyterminus of the modifier protein and a free amino group of the targetprotein. In another embodiment, a target protein is specific to a classof modifier proteins.

[0039] For example, the target protein of the present invention may becullin proteins such as Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5, whichare known to be the target proteins of Nedd8. In one embodiment, thetarget protein of the present invention includes any protein havingubiquitin ligase activity or is part of a protein complex havingubiquitin ligase activity. In another embodiment, the target protein ofthe present invention includes any protein to which ubiquitin conjugatesfor processing or degradation, e.g., p53, IκB, NF-κB, β-adrenergicreceptor, cyclin E, p27^(Kip1), etc.

[0040] According to one embodiment of the present invention, the targetprotein used in the screening assays provided by the present inventioncan be any protein that produces a detectable signal upon deconjugation,removal, or separation from its modifier protein. Such detectable signalcan be any assayable signal including, without limitation, enzymatic,spectroscopic, fluorescent, or functional signals, or a signal producedupon specific molecular interaction. For example, the target protein canbe an enzyme such as peroxidase, alkaline phosphatase, and luciferase.The target protein can also be a fluorescent protein obtained viaprotein modification or a naturally fluorescent protein including,without limitation green fluorescent protein, yellow fluorescentprotein, cyan fluorescent protein, dsRed, and the derivatives thereof.

[0041] In one embodiment, the target protein of the present invention isused in the screening assays of the present invention with a 26Sproteasome and may be any protein that can structurally fit or refoldwithin the inner chamber of a 20S proteasome. Normally the inner chamberof a 20S proteasome can structurally accommodate any protein that is 70kD or less.

[0042] According to the present invention, a target protein isdeconjugated, removed, or separated from its modifier protein by beingexposed or contacted to a polypeptide having the JAB subunit or JAMdomain. Such exposure or contact may be in vivo via administering thepolypeptide to a subject in need of such treatment or in vitro viaincubating the polypeptide with the target protein. In one embodiment,an inhibitor of a polypeptide having the JAB subunit or JAM domain canbe used to increase the association of a modifier protein to a targetprotein in vitro or in vivo.

[0043] According to another aspect of the invention, a polypeptidehaving the JAB subunit or JAM domain can be used for screening assaysfor agents that affect the deconjugation, removal, or separation of amodifier protein from a target protein. The screening assays provided bythe present invention can be carried out by incubating in the presenceand absence of a test agent, a target protein, e.g., conjugated with amodifier protein and a polypeptide containing the JAB subunit or JAMdomain, and determining the effect of the test agent.

[0044] Normally an increase or decrease in the amount of the targetprotein not conjugated to the modifier protein caused by the test agentis indicative of an agent capable of affecting deconjugation, removal,or separation of the modifier protein from the target protein. Forexample, a test agent decreasing the amount of the target protein notconjugated to the modifier protein is indicative of an agent decreasingthe deconjugation of the modifier protein from the target protein.

[0045] In one embodiment, the polypeptide used in the screening assaysprovided by the present invention is a polypeptide complex of 26Sproteasome while the modifier protein is ubiquitin. The polypeptidecomplex of 26S proteasome can be obtained by any suitable meansavailable in the art. For example, 26S proteasome can be purified fromeukaryotic cells or tissues, e.g., S. cerevisiae or human.

[0046] In another embodiment, the incubation in the presence and absenceof a test agent, a target protein, and 26S proteasome of the screeningassays provided by the present invention is carried out in the presenceof a 20S inhibitor or an inhibitor of the degradation process associatedwith the de-ubiquitination process of the 26S proteasome pathway. Any20S inhibitor or inhibitor of the degradation process can be used forthe purpose of the present invention. For example, a 20S inhibitor canbe MG132, lactacystin, epoxomycin, PS-349, PS-519, LLnL, or thederivatives thereof. In general, such inhibitor prevents or decreasesthe degradation of a target protein that is not conjugated to a modifierprotein, e.g., ubiquitin.

[0047] In yet another embodiment, the incubation is conducted further inthe presence of an energy source, e.g., ATP. In still anotherembodiment, the incubation is conducted further in the presence of aninhibitor of deubiquitination by a conventional ubiquitin isopeptidase,e.g., an ubiquitin isopeptidase other than those that associated with aJAB subunit such as 26S proteasome. One example of such inhibitor isubiquitin aldehyde, e.g., at 2-5 μM.

[0048] The test agent used for the screening methods of the presentinvention can be any agent from any library of compounds or molecules.In one embodiment, the test agent is selected or derived from compoundslikely to inhibit the activity of metalloproteinase, e.g., compoundshaving zinc-binding functionality. For example, the test agent can beany compounds having a hydroxamate moiety or a member of a hydroxamatecompound library, reverse hydroxamate compound library, thiol compoundlibrary, carboxylate compound library, or phosphonic acid compoundlibrary.

[0049] According to another aspect of the present invention, the JAMdomain can be used for rational drug design or as a guide foridentifying agents that are capable of affecting the activity of the JAMdomain or any polypeptide containing the JAM domain, e.g., identifyinhibitors of the JAM domain. In one embodiment, the structurecoordinates or atomic coordinates of the JAM domain or any polypeptidecontaining the JAM domain are used to design a potential inhibitor thatwill form a covalent or non-covalent bond with one or more amino acidswithin the JAM domain or metal ions bound by the JAM domain. In anotherembodiment, the potential inhibitor is designed to form a covalent bondor non-covalent bond with histidine or aspartate of the JAM domain. Suchdesigned potential inhibitor can be synthesized by any suitable meansand be tested for their ability to inhibit the activity, e.g., peptidaseactivity of the JAM domain.

[0050] The structure or atomic coordinates of the JAM domain or apolypeptide containing the JAM domain refer to mathematical coordinatesderived from mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) of the JAM domain or the polypeptide containing the JAM domainin crystal form. The diffraction data normally are used to calculate anelectron density map of the repeating unit of the crystal. The electrondensity maps are generally used to establish the positions of theindividual atoms within the unit cell of the crystal.

[0051] Various methods can be used to obtain the structure or atomiccoordinates of the JAM domain or a polypeptide containing the JAMdomain. For example, three dimensional diffraction data for apolypeptide containing the JAM domain can be collected at temperaturesranging from 100-274 K using an area detector and radiation from arotating-anode X-ray generator and from the Stanford synchrotron. Thesedata, along with data collected from a heavy atom derivative of thepolypeptide, can be processed and the structure can be solved by methodswhich make use of the isomorphous differences between a derivative andnative polypeptide and/or make use of the anomalous X-ray scatteringfrom the heavy atom in the derivative. In one embodiment, the structureor atomic coordinates of one JAM containing polypeptide can be solved byusing the phases of another JAM containing polypeptide structure orsections thereof that has already been previously determined. Highresolution data sets can be solved by direct methods.

[0052] According to another aspect of the present invention, any agentthat is capable of inhibiting or decreasing the deconjugation, removal,or separation of a modifier protein from a target protein can be usedtherapeutically to treat various conditions associated with proteinregulation, e.g., de-ubiquitination or de-neddylation. For example, anyagent identified by the screening methods of the present invention thatis able to decrease the deconjugation of a modifier protein from atarget protein, e.g., an inhibitor of the isopeptidase activity of 26Sproteasome can be used therapeutically to treat neoplastic growth,angiogenesis, infection, chronic inflammation, asthma, ischemia andreperfusion injury, multiple sclerosis, rheumatoid arthritis, andpsoriasis.

[0053] The agents of the present invention useful for therapeutictreatment can be administered alone, in a composition with a suitablepharmaceutical carrier, or in combination with other therapeutic agents.An effective amount of the agents to be administered can be determinedon a case-by-case basis. Factors should be considered usually includeage, body weight, stage of the condition, other disease conditions,duration of the treatment, and the response to the initial treatment.Typically, the agents are prepared as an injectable, either as a liquidsolution or suspension. However, solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The agent can also be formulated into an enteric-coated tabletor gel capsule according to known methods in the art. The agents of thepresent invention may be administered in any way which is medicallyacceptable which may depend on the disease condition or injury beingtreated. Possible administration routes include injections, byparenteral routes such as intravascular, intravenous, intraepidural orothers, as well as oral, nasal, ophthalmic, rectal, topical, orpulmonary, e.g., by inhalation. The agents may also be directly appliedto tissue surfaces, e.g., during surgery. Sustained releaseadministration is also specifically included in the invention, by suchmeans as depot injections or erodible implants.

EXAMPLES

[0054] The following examples are intended to illustrate but not tolimit the invention in any manner, shape, or form, either explicitly orimplicitly. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used.

Example 1

[0055] De-Ubiquination by 26S Proteasome

[0056] To demonstrate that an isopeptidase activity associated with the26S proteasome can deubiquitinate a target polypeptide in vitro,experiments were conducted to incubate multiubiquitinated Sic1 substrate(300 nM) for 0 or 5 minutes at 25° C. in the presence of purified 26Sproteasome (100 nM), epoxomicin (100 μM), and ubiquitin aldehyde (5 μM).

[0057] Epoxomicin was included to uncouple the deubiquitination anddegradation of a substrate by the 26S proteasome, since these twoprocesses are normally tightly coupled. Epoxomicin forms a covalentadduct with the catalytically active N-terminal threonine residues ofbeta subunits of the 20S proteasome, thereby eliminating the proteolyticactivity of this particle. At the end of the incubation, samples weresupplemented with SDS-PAGE sample buffer, fractionated on anSDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblottedwith antibodies directed against Sic1. Filter-bound antibodies weredecorated with goat-anti-rabbit antibody conjugated to horse radishperoxidase and visualized with ECL reagents.

[0058] This experiment demonstrated that inhibition of 20S peptidaseactivity is required to observe the production of deubiquitinated Siclby 26S proteasome. In the absence of epoxomicin, ubiquitinated Sic1 wascompletely degraded by the 26S proteasome . In the presence ofepoxomicin, the Sic1 was not degraded, but the majority of it wasdeubiquitinated such that the input substrate was converted from aheterogeneous smear of >220 KD to a discrete species of ˜40 KD, whichcorresponds to SicI lacking any covalently attached ubiquitin molecules.

[0059] To test if this reaction is dependent upon ATP,multiubiquitinated Sic 1 substrate (300 nM) and purified 26S proteasome(100 nM) were incubated in the presence or absence of epoxomicin (100μM), glucose (30 mM) plus hexokinase (5 U/ml), apyrase (15 U/ml), andubiquitin vinyl sulfone (Ub-PVS; 2.5 μM) as indicated. This experimentrevealed that ATP is required to observe the deubiquitination of targetpolypeptide by purified 26S proteasome. Specifically, in the presence ofmultiubiquitinated Sic 1 substrate, 26S proteasome (which contains ATP),and epoxomicin, the input substrate was converted from a heterogeneoussmear of >220 KD to a discrete species of ˜40 KD, which corresponds toSic1 lacking any covalently attached ubiquitin molecules. However, inthe additional presence of apyrase or glucose plus hexokinase (both ofwhich consume ATP), no deubiquitination of the multiubiquitinated Sic 1substrate was observed. This reaction was not sensitive to ubiquitinvinyl sulfone, which is an inhibitor of conventional ubiquitinisopeptidases.

[0060] These data demonstrate that the 26S proteasome recognizes aubiquitinated substrate, and begins to translocate it into the internalcavity of the 20S proteasome, where it is degraded. As the substrate isbeing translocated into the 20S proteasome, the ubiquitin chains areclipped off by a previously unknown isopeptidase that is in the 26Sproteasome. Under normal circumstances this is not detected becausesubstrate degradation and removal of the ubiquitin chains occurscontemporaneously. However, in the presence of an inhibitor of the 20Speptidases, the substrate is completely deubiquitinated and translocatedinto the central cavity of the 20S proteasome, but is not degraded.Hence the deubiquitinated product is readily detected

[0061] Mass spectrometric analysis of affinity-purified yeast 26Sproteasome that is active in deubiquitinating Sic 1 revealed only asingle deubiquitinating enzyme: Ubp6 (Verma et al., Mol Biol Cell11:3425 (2000)). However, 26S proteasome purified from a ubp6Δ mutantyeast strain and assayed as described above was fully competent todeubiquitinate multiubiquitinated Sic1 to yield Sic1 lacking anycovalently attached ubiquitin molecules..

[0062] The above demonstrations enable an assay wherein the substrate isobserved to be converted from a ubiquitinated molecule to adeubiquitinated molecule. There are many straightforward procedures bywhich this conversion can be monitored. For example, the removal of amultiubiquitin chain from a protein can change enzymatic, spectroscopic,fluorescent, or molecular interaction properties of the ubiquitinatedprotein. As one specific example, a protein that emits light e.g.,luciferase will be made inactive due to the attachment of multiubiquitinchains.

[0063] The multiubiquitin chains will be attached by enzymatic,chemical, molecular genetic, or a combination of these methods. Thecleavage of ubiquitin chains from the inactive luciferase substrate asit is being translocated into the inner cavity of the 20S proteasome inthe presence of a 20S peptidase inhibitor will enable the luciferase tofold and thereby acquire the ability to emit light upon ATP hydrolysis.

[0064] The inner chamber of the 20S proteasome can accommodate a foldedprotein of approximately 70 kD. Therefore, any protein domain less than70 kD whose enzymatic, spectroscopic, fluorescent, or molecularinteraction properties can be reversibly altered by attachment ofubiquitin or a multiubiquitin chain can be used as a substrate tomonitor ubiquitin isopeptidase activity of the 26S proteasome based onthe methods that are disclosed herein.

[0065] Inhibition of the 26S proteasome's ability to degrade proteins byblockage of ubiquitin isopeptidase activity would be applicable to thetreatment of cancer, ischemia and reperfusion injury, and diseasescharacterized by excessive inflammation or autoimmune responses,including asthma, rheumatoid arthritis, psoriasis, and multiplesclerosis.

[0066] To test whether the deconjugation of ubiquitin from a substrateprotein is required for its degradation, we evaluated the degradation ofan ubiquitin-proteasome pathway substrate in cells deficient inRpn11-asssociated metalloprotease activity. A set of four congenicstrains was generated: MPR (wild type), mpr1-1, mpr1-1 harboring anintegrated copy of RPN11 in which the codons for histidine 109 and 111have been mutated to alanine (mpr1-1 leu2::LEU2-rpn11AxA), and mpr1-1harboring an integrated copy of wild type RPN11 (mpr1-1leu2::LEU2-RPN11). mpr1-1 is a temperature-sensitive allele of RPN11,and complementation tests demonstrated that wild type RPN11 but notrpn11AxA was able to complement the temperature-sensitive growth defectof mpr1-1.

[0067] All of the strains described above were transformed with aplasmid that encodes UbV⁷⁶Val-e^(ΔK)-βGa1, which is an unstable proteinthat is degraded via the ubiquitin-proteasome pathway. All strains werepulse-radiolabeled with ³⁵S-Translabel for 5 min at 37° C., and then attime 0 a chase was initiated by addition of 10 mM cold methionine and500 μg/ml cycloheximide. At 7, 14, and 22 minutes following theinitiation of chase, aliquots of the culture were removed and processedfor immunoprecipitation with antibodies directed againstβ-galactosidase. Immunoprecipitated proteins were fractionated bySDS-PAGE and visualized by autoradiography.

[0068] The data demonstrated that the normally unstableUb^(V76)Val-e^(ΔK)-βGa1 reporter was dramatically stabilized in mpr1-1cells. Rapid turnover of the test protein in mrp1-1 cells was restoredby an integrated copy of RPN11, but not rpn11AxA. Thus, themetalloprotease active site of Rpn11 was required for rapid turnover ofproteins by the ubiquitin-proteasome pathway in vivo.

[0069] This result was further confirmed in vitro. We demonstrated thatpurified 26S proteasomes that lack critical metalloprotease active siteresidues of Rpn11 were unable to deubiquitinate and degradeubiquitinated Sic1. 26S proteasomes were affinity-purified fromSaccharomyces cerevisiae cells in which the PRE1 gene was modified toencode a Pre1 polypeptide tagged with a FLAG epitope, as described(Verma et al., 2000 Mol Biol Cell 11:3425).

[0070] Wild type 26S proteasome was purified from a strain with thegenotype

[0071] pre1::PRE1-FLAG-HIS6 (URA3) his3-11 ade2-1 112 trp1-Δ2 can1-100,mpr1-1,

[0072] leu2:.LEU2-RPN11, and mutant 26S proteasome was purified from astrain with the

[0073] genotype pre1::PRE1-FLAG-HIS6 (URA3) his3-11 ade2-1 112 trp1-Δ2can1-100, mpr1-1,

[0074] leu2::LEU2-rpn11AXA.

[0075] Purified proteasomes were evaluated by immunoblotting withantibodies directed against Schizosaccharomyces pombe Pad1, whichcross-reacted with Rpn11. Wild type and point mutant Rpn11 polypeptideswere readily distinguished from Mpr1-1 polypeptide, because the latteris truncated due to a frameshift mutation. The immunoblot analysisrevealed that both wild type and mutant proteasomes contained onlyfull-length Rpn11polypeptide, and none of the truncated Mpr1-1polypeptide was detected. Thus, the results we obtained are directlyattributable to the properties of the Rpn11 and Rpn11AxA proteins sinceno Mpr1-1 protein is present. Mass spectrometric analysis confirmed thatthe wild type and mutant proteasomes were of equal composition, and allsubunits were present.

[0076] To evaluate their activity, wild type and Rpn11AxA mutant 26Sproteasomes (100 nM) were incubated with ubiquitinated Sic 1 (300nM)plus ATP (2mM) in the presence or absence of 100 μM epoxomicin for 0 or5 minutes at 25° C., as indicated. Reactions were terminated by theaddition of SDS-PAGE sample buffer, fractionated by SDS-PAGE,transferred to nitrocellulose, and immunoblotted with antibodiesdirected against Sic1. Filter-bound antibodies were decorated withgoat-anti-rabbit antibody conjugated to horse radish peroxidase andvisualized with ECL reagents. The data demonstrated that mutantproteasomes were unable to degrade multiubiquitinated Sic1 even in theabsence of epoxomicin, and were unable to deubiquitinatemultiubiquitinated Sic1 (ie to convert multiubiquitinated Sic1 from aheterogeneous smear of >220 KD to a discrete species of ˜40 KD, whichcorresponds to Sic1 lacking any covalently attached ubiquitin molecules)in the presence of epoxomicin.

Example 2

[0077] De-Neddylation By COP9 Signalsome (CSN)

[0078] To identify a putative active site within the CSN complex thatmight mediate its ability to promote the cleavage of Nedd8-cullinconjugates, we subjected all eight known subunits of the CSN to sequenceanalysis by computer. Detailed inspection revealed that the Jab1 subunitcontains conserved histidines and a conserved aspartic acid that arereminiscent of zinc-coordinating residues in a metallo-beta-lactamase.An alignment of these residues is presented in FIG. 3.

[0079] On the basis of this analysis, we hypothesized that CSNrepresents the founding member of a novel class of metalloproteases. Totest this hypothesis, we performed the following experiments. First, weevaluated the effect of a divalent cation chelator on the Nedd8conjugate cleavage activity associated with CSN. Mutant csn5Δ S. pombecell extract that contained neddylated Pcu1 was incubated with either noadditions (−) or following addition (+) of wild type lysate (whichcontained active CSN) supplemented with either methanol vehicle (MeOH),1 mM 1,10-phenanthroline (O-PT), or 1 mM, 10 mM, or 20 mM EDTA.

[0080] Reactions were incubated 30 minutes at 30° C. and terminated byaddition of SDS-PAGE sample buffer, fractionated on anSDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblottedwith antibodies directed against S. pombe Cul1. Filter-bound antibodieswere decorated with goat-anti-rabbit antibody conjugated to horse radishperoxidase and visualized with ECL reagents (Lyapina, S., et al.,Science 18: 1382-5 (2001)).

[0081] The data demonstrated that no inhibition was seen with 1 mM EDTA,and less than 50% inhibition was seen with 10 mM EDTA. However, 20 mMEDTA almost completely inhibited the reaction. In the context of thisdescription, ‘inhibition’ means that Nedd8 was not deconjugated fromCul1 upon addition of CSN, whereas ‘no inhibition’ means that Nedd8 wasefficiently deconjugated from Cul1 upon addition of CSN. The status ofNedd8 conjugation to Cul1 was determined by the mobility of Cul1 on theSDS-polyacrylamide gel. Nedd8-conjugated Cul1 migrated more slowly thanunmodified Cul.

[0082] It is known that some metal-dependent enzymes are relativelyinsensitive to inhibition by EDTA, but are nevertheless potentlyinhibited by the chelator 1,10-phenanthroline. Intriguingly,1,10-phenanthroline at 1 mM completeley inhibited the Nedd8 conjugatecleavage activity of CSN. Thus, biochemical data suggested that CSN isin fact a metalloprotease.

[0083] To further confirm the hypothesis that arose from our sequenceanalysis, we individually mutated the conserved histidines and asparticacid in Schizosaccharomyces pombe csn5⁺, and tested the ability of eachpoint-mutated gene to complement the cul1in deneddylation defect of acsn5Δ mutant (Lyapina, S., et al., Science 18:1382-5 (2001); Zhou, C.,et al., BMC Biochemistry 2:7 (2001)). Wild type S. pombe csn5⁺ and theindicated mutant derivatives were inserted into the S. pombe expressionvector pREP41 and transfected into S. pombe csn5Δ cells.

[0084] Transformants were evaluated for modification state of Pcu1 byfractionating cell extracts on an SDS-polyacrylamide gel, transferringfractionated proteins to nitrocellulose, and immunoblotting withantibodies directed against S. pombe Cul1. Filter-bound antibodies weredecorated with goat-anti-rabbit antibody conjugated to horse radishperoxidase and visualized with ECL reagents.

[0085] The data from this experiment demonstrated that csn5Δ cells thatcontained empty vector accumulated Pcul exclusively in theNedd8-modified form. This accumulation was reversed upon expression ofwild type csn5⁺, but not the H118A, H120A, and D131N mutants. Thus, theputative active site residues identified by computer analysis areabsolutely required for CSN activity in deconjugating Nedd8 from Cul1 invivo.

[0086] Based on the data described above, we conclude that CSN is anovel metalloprotease. The active site of CSN resides, at least in part,in the Jab1/Csn5 subunit, and this active site is proposed to bind ametal ion which initiates hydrolytic attack of the isopeptide bondscleaved by CSN. These findings enable the development of targetedscreens for compounds that inhibit or enhance the catalytic activity ofCSN, and also allow for the rationale design of compounds that modulateCSN activity based on knowledge of the mechanism of action of othermetalloenzymes.

[0087] Finally, this knowledge might be used for two further purposes.First, we suggest it should be possible to re-engineer the specificityof CSN, Jab1, or related proteins to create novel isopeptidases withdesired specificities. Such enzymes might be useful in a variety ofapplications involving the cleavage of protein cross-links orconjugates. Second, it should be possible to isolate or design compoundsthat inhibit the prokaryotic homologs of Csn5/Jab1. These inhibitors mayhave a number of applications, including use as antibiotics.

[0088] We have also tested the role of Cys box (Cys 145) in associationwith CSN's ability to deconjugate Nedd8 from Cul1, using an in vitrodeconjugation assay as described above. The experimental datademonstrate that a Jab1 mutant in which the conserved cysteine of theCys box (Cys 145) is mutated to alanine has wild-type levels of Nedd8conjugate cleavage activity.

Example 3

[0089] AMSH, AMSH1, and AMSH2

[0090] As discussed above, we have documented the existence of a novelmetalloprotease active site motif that we have dubbed the JAM domain(for Jab1 -associated metalloenzyme motif). The Rpn11 subunit of the 26Sproteosome and the Csn5 subunit of the COP9-signalosome (CSN) bothcontain a JAM domain. We have provided direct evidence that the JAMdomain of Csn5 is essential for the Nedd8 isopeptidase activity of CSN,and the JAM domain of Rpn11 is essential for the ubiquitin isopeptidaseactivity of the 26S proteosome.

[0091] Our data teach that eukaryotic JAM domain proteins containisopeptidase activity that deconjugate modifier proteins from targetproteins, wherein the carboxy terminus of the modifier protein isattached via a peptide bond to a free amino group of the target protein,including but not restricted to the amino terminus and the epsilon aminogroup of lysine residues in the target protein.

[0092] In addition to Csn5 and Rpn11, there are many proteins expressedin human cells that contain an intact JAM domain. (see Table 1) The AMSHproteins have been implicated in cytokine signaling, TGF-b signaling,and survival of hippocampal neurons (Ishii, N., et al., Mol Cell Biol.24:8626-37 (2001); Itoh, F., et al., EMBO J. 15:4132-42 (2001); Tanaka,N., et al., J Biol Chem. 27:19129-35 (1999)).

[0093] Our data teach that the histidine 335, histidine 337, andaspartate 348 residues of AMSH (and the equivalent residues in AMSH1 andAMSH2, see FIG. 2) specify a metalloprotease active site. Furthermore,our data teach that compounds that inhibit the active site specified bythese residues will inhibit the ability of AMSH, AMSH1, and AMSH2 todeconjugate a modifier protein from a target protein, wherein themodifier protein includes NEDD8, UBL1, SMT3H2, SMT3H1, APG12, FAT10,Fau, UCRP/ISG15, URM1, or UBL5.

[0094] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1 22 1 14 PRT Artificial sequence JAM domain 1 His Xaa His Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Asp 1 5 10 2 17 PRT Artificial sequence JAMdomain 2 Gly Trp Xaa His Xaa His Pro Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa1 5 10 15 Asp 3 246 PRT Homo sapiens 3 Thr Met Ile Ile Met Asp Ser PheAla Leu Pro Val Glu Gly Thr Glu 1 5 10 15 Thr Arg Val Asn Ala Gln AlaAla Ala Tyr Glu Tyr Met Ala Ala Tyr 20 25 30 Ile Glu Asn Ala Lys Gln ValGly Arg Leu Glu Asn Ala Ile Gly Trp 35 40 45 Tyr His Ser His Pro Gly TyrGly Cys Trp Leu Ser Gly Ile Asp Val 50 55 60 Ser Thr Gln Met Leu Asn GlnGln Phe Gln Glu Pro Phe Val Ala Val 65 70 75 80 Val Ile Asp Pro Thr ArgThr Ile Ser Ala Gly Lys Val Asn Leu Gly 85 90 95 Ala Phe Arg Thr Tyr ProLys Gly Tyr Lys Pro Pro Asp Glu Gly Pro 100 105 110 Ser Glu Tyr Gln ThrIle Pro Leu Asn Lys Ile Glu Asp Phe Gly Val 115 120 125 His Cys Lys GlnTyr Tyr Ala Leu Glu Val Ser Tyr Phe Lys Ser Ser 130 135 140 Leu Asp ArgLys Leu Leu Glu Leu Leu Trp Asn Lys Tyr Trp Val Asn 145 150 155 160 ThrLeu Ser Ser Ser Ser Leu Leu Thr Asn Ala Asp Tyr Thr Thr Gly 165 170 175Gln Val Phe Asp Leu Ser Glu Lys Leu Glu Gln Ser Glu Ala Gln Leu 180 185190 Gly Arg Gly Ser Phe Met Leu Gly Leu Glu Thr His Asp Arg Lys Ser 195200 205 Glu Asp Lys Leu Ala Lys Ala Thr Arg Asp Ser Cys Lys Thr Thr Ile210 215 220 Glu Ala Ile His Gly Leu Met Ser Gln Val Ile Lys Asp Lys LeuPhe 225 230 235 240 Asn Gln Ile Asn Ile Ser 245 4 245 PRT Homo sapiens 4Thr Val Arg Val Ile Asp Val Phe Ala Met Pro Gln Ser Gly Thr Gly 1 5 1015 Val Ser Val Glu Ala Val Asp Pro Val Phe Gln Ala Lys Met Leu Asp 20 2530 Met Leu Lys Gln Thr Gly Arg Pro Glu Met Val Val Gly Trp Tyr His 35 4045 Ser His Pro Gly Phe Gly Cys Trp Leu Ser Gly Val Asp Ile Asn Thr 50 5560 Gln Gln Ser Phe Glu Ala Leu Ser Glu Arg Ala Val Ala Val Val Val 65 7075 80 Asp Pro Ile Gln Ser Val Lys Gly Lys Val Val Ile Asp Ala Phe Arg 8590 95 Leu Ile Asn Ala Asn Met Met Val Leu Gly His Glu Pro Arg Gln Thr100 105 110 Thr Ser Asn Leu Gly His Leu Asn Lys Pro Ser Ile Gln Ala LeuIle 115 120 125 His Gly Leu Asn Arg His Tyr Tyr Ser Ile Thr Ile Asn TyrArg Lys 130 135 140 Asn Glu Leu Glu Gln Lys Met Leu Leu Asn Leu His LysLys Ser Trp 145 150 155 160 Met Glu Gly Leu Thr Leu Gln Asp Tyr Ser GluHis Cys Lys His Asn 165 170 175 Glu Ser Val Val Lys Glu Met Leu Glu LeuAla Lys Asn Tyr Asn Lys 180 185 190 Ala Val Glu Glu Glu Asp Lys Met ThrPro Glu Gln Leu Ala Ile Lys 195 200 205 Asn Val Gly Lys Gln Asp Pro LysArg His Leu Glu Glu His Val Asp 210 215 220 Val Leu Met Thr Ser Asn IleVal Gln Cys Leu Ala Ala Met Leu Asp 225 230 235 240 Thr Val Val Phe Lys245 5 421 PRT Homo sapiens 5 Met Pro Asp His Thr Asp Val Ser Leu Ser ProGlu Glu Arg Val Arg 1 5 10 15 Ala Leu Ser Lys Leu Gly Cys Asn Ile ThrIle Ser Glu Asp Ile Thr 20 25 30 Pro Arg Arg Tyr Phe Arg Ser Gly Val GluMet Glu Arg Met Ala Ser 35 40 45 Val Tyr Leu Glu Glu Gly Asn Leu Glu AsnAla Phe Val Leu Tyr Asn 50 55 60 Lys Phe Ile Thr Leu Phe Val Glu Lys LeuPro Asn His Arg Asp Tyr 65 70 75 80 Gln Gln Cys Ala Val Pro Glu Lys GlnAsp Ile Met Lys Lys Leu Lys 85 90 95 Glu Ile Ala Phe Pro Arg Thr Asp GluLeu Lys Asn Asp Leu Leu Lys 100 105 110 Lys Tyr Asn Val Glu Tyr Gln GluTyr Leu Gln Ser Lys Asn Lys Tyr 115 120 125 Lys Ala Glu Ile Leu Lys LysLeu Glu His Gln Arg Leu Ile Glu Ala 130 135 140 Glu Arg Lys Arg Ile AlaGln Met Arg Gln Gln Gln Leu Glu Ser Glu 145 150 155 160 Gln Phe Leu PhePhe Glu Asp Gln Leu Lys Lys Gln Glu Leu Ala Arg 165 170 175 Gly Gln MetArg Ser Gln Gln Thr Ser Gly Leu Ser Glu Gln Ile Asp 180 185 190 Gly SerAla Leu Ser Cys Phe Ser Thr His Gln Asn Asn Ser Leu Leu 195 200 205 AsnVal Phe Ala Asp Gln Pro Asn Lys Ser Asp Ala Thr Asn Tyr Ala 210 215 220Ser His Ser Pro Pro Val Asn Arg Ala Leu Thr Pro Ala Ala Thr Leu 225 230235 240 Ser Ala Val Gln Asn Leu Val Val Glu Gly Leu Arg Cys Val Val Leu245 250 255 Pro Glu Asp Leu Cys His Lys Phe Leu Gln Leu Ala Glu Ser AsnThr 260 265 270 Val Arg Gly Ile Glu Thr Cys Gly Ile Leu Cys Gly Lys LeuThr His 275 280 285 Asn Glu Phe Thr Ile Thr His Val Ile Val Pro Lys GlnSer Ala Gly 290 295 300 Pro Asp Tyr Cys Asp Met Glu Asn Val Glu Glu LeuPhe Asn Val Gln 305 310 315 320 Asp Gln His Asp Leu Leu Thr Leu Gly TrpIle His Thr His Pro Thr 325 330 335 Gln Thr Ala Phe Leu Ser Ser Val AspLeu His Thr His Cys Ser Tyr 340 345 350 Gln Leu Met Leu Pro Glu Ala IleAla Ile Val Cys Ser Pro Lys His 355 360 365 Lys Asp Thr Gly Ile Phe ArgLeu Thr Asn Ala Gly Met Leu Glu Val 370 375 380 Ser Ala Cys Lys Lys LysGly Phe His Pro His Thr Lys Glu Pro Arg 385 390 395 400 Leu Phe Ser IleCys Lys His Val Leu Val Lys Asp Ile Lys Ile Ile 405 410 415 Val Leu AspLeu Arg 420 6 461 PRT Homo sapiens 6 Met Asp Gln Pro Phe Thr Val Asn SerLeu Lys Lys Leu Ala Ala Met 1 5 10 15 Pro Asp His Thr Asp Val Ser LeuSer Pro Glu Glu Arg Val Arg Ala 20 25 30 Leu Ser Lys Leu Gly Cys Asn IleThr Ile Ser Glu Asp Ile Thr Pro 35 40 45 Arg Arg Tyr Phe Arg Ser Gly ValGlu Met Glu Arg Met Ala Ser Val 50 55 60 Tyr Leu Glu Glu Gly Asn Leu GluAsn Ala Phe Val Leu Tyr Asn Lys 65 70 75 80 Phe Ile Thr Leu Phe Val GluLys Leu Pro Asn His Arg Asp Tyr Gln 85 90 95 Gln Cys Ala Val Pro Glu LysGln Asp Ile Met Lys Lys Leu Lys Glu 100 105 110 Ile Ala Phe Pro Arg ThrAsp Glu Leu Lys Asn Asp Leu Leu Lys Lys 115 120 125 Tyr Asn Val Glu TyrGln Glu Tyr Leu Gln Ser Lys Asn Lys Tyr Lys 130 135 140 Ala Glu Ile LeuLys Lys Leu Glu His Gln Arg Leu Ile Glu Ala Glu 145 150 155 160 Arg LysArg Ile Ala Gln Met Arg Gln Gln Gln Leu Glu Ser Glu Gln 165 170 175 PheLeu Phe Phe Glu Asp Gln Leu Lys Lys Gln Glu Leu Ala Arg Gly 180 185 190Gln Met Arg Ser Gln Gln Thr Ser Gly Leu Ser Glu Gln Ile Asp Gly 195 200205 Ser Ala Leu Ser Cys Phe Ser Thr His Gln Asn Asn Ser Leu Leu Asn 210215 220 Val Phe Ala Asp Gln Pro Asn Lys Ser Asp Ala Thr Asn Tyr Ala Ser225 230 235 240 His Ser Pro Pro Val Asn Arg Ala Leu Thr Pro Ala Ala ThrLeu Ser 245 250 255 Ala Val Gln Asn Leu Val Val Glu Gly Leu Arg Cys ValVal Leu Pro 260 265 270 Glu Asp Leu Cys His Lys Phe Leu Gln Leu Ala GluSer Asn Thr Val 275 280 285 Arg Gly Ile Glu Thr Cys Gly Ile Leu Cys GlyLys Leu Thr His Asn 290 295 300 Glu Phe Thr Ile Thr His Val Ile Val ProLys Gln Ser Ala Gly Pro 305 310 315 320 Asp Tyr Cys Asp Met Glu Asn ValGlu Glu Leu Phe Asn Val Gln Asp 325 330 335 Gln His Asp Leu Leu Thr LeuGly Trp Ile His Thr His Pro Thr Gln 340 345 350 Thr Ala Phe Leu Ser SerVal Asp Leu His Thr His Cys Ser Tyr Gln 355 360 365 Leu Met Leu Pro GluAla Ile Ala Ile Val Cys Ser Pro Lys His Lys 370 375 380 Asp Thr Gly IlePhe Arg Leu Thr Asn Ala Gly Met Leu Glu Val Ser 385 390 395 400 Ala CysLys Lys Lys Gly Phe His Pro His Thr Lys Glu Pro Arg Leu 405 410 415 PheSer Ile Gln Lys Phe Leu Ser Gly Ile Ile Ser Gly Thr Ala Leu 420 425 430Glu Met Glu Pro Leu Lys Ile Gly Tyr Gly Pro Asn Gly Phe Pro Leu 435 440445 Leu Gly Ile Ser Arg Ser Ser Ser Pro Ser Glu Gln Leu 450 455 460 7424 PRT Homo sapiens 7 Met Ser Asp His Gly Asp Val Ser Leu Pro Pro GluAsp Arg Val Arg 1 5 10 15 Ala Leu Ser Gln Leu Gly Ser Ala Val Glu ValAsn Glu Asp Ile Pro 20 25 30 Pro Arg Arg Tyr Phe Arg Ser Gly Val Glu IleIle Arg Met Ala Ser 35 40 45 Ile Tyr Ser Glu Glu Gly Asn Ile Glu His AlaPhe Ile Leu Tyr Asn 50 55 60 Lys Tyr Ile Thr Leu Phe Ile Glu Lys Leu ProLys His Arg Asp Tyr 65 70 75 80 Lys Ser Ala Val Ile Pro Glu Lys Lys AspThr Val Lys Lys Leu Lys 85 90 95 Glu Ile Ala Phe Pro Lys Ala Glu Glu LeuLys Ala Glu Leu Leu Lys 100 105 110 Arg Tyr Thr Lys Glu Tyr Thr Glu TyrAsn Glu Glu Lys Lys Lys Glu 115 120 125 Ala Glu Glu Leu Ala Arg Asn MetAla Ile Gln Gln Glu Leu Glu Lys 130 135 140 Glu Lys Gln Arg Val Ala GlnGln Lys Gln Gln Gln Leu Glu Gln Glu 145 150 155 160 Gln Phe His Ala PheGlu Glu Met Ile Arg Asn Gln Glu Leu Glu Lys 165 170 175 Glu Arg Leu LysIle Val Gln Glu Phe Gly Lys Val Asp Pro Gly Leu 180 185 190 Gly Gly ProLeu Val Pro Asp Leu Glu Lys Pro Ser Leu Asp Val Phe 195 200 205 Pro ThrLeu Thr Val Ser Ser Ile Gln Pro Ser Asp Cys His Thr Thr 210 215 220 ValArg Pro Ala Lys Pro Pro Val Val Asp Arg Ser Leu Lys Pro Gly 225 230 235240 Ala Leu Ser Asn Ser Glu Ser Ile Pro Thr Ile Asp Gly Leu Arg His 245250 255 Val Val Val Pro Gly Arg Leu Cys Pro Gln Phe Leu Gln Leu Ala Ser260 265 270 Ala Asn Thr Ala Arg Gly Val Glu Thr Cys Gly Ile Leu Cys GlyLys 275 280 285 Leu Met Arg Asn Glu Phe Thr Ile Thr His Val Leu Ile ProLys Gln 290 295 300 Ser Ala Gly Ser Asp Tyr Cys Asn Thr Glu Asn Glu GluGlu Leu Phe 305 310 315 320 Leu Ile Gln Asp Gln Gln Gly Leu Ile Thr LeuGly Trp Ile His Thr 325 330 335 His Pro Thr Gln Thr Ala Phe Leu Ser SerVal Asp Leu His Thr His 340 345 350 Cys Ser Tyr Gln Met Met Leu Pro GluSer Val Ala Ile Val Cys Ser 355 360 365 Pro Lys Phe Gln Glu Thr Gly PhePhe Lys Leu Thr Asp His Gly Leu 370 375 380 Glu Glu Ile Ser Ser Cys ArgGln Lys Gly Phe His Pro His Ser Lys 385 390 395 400 Asp Pro Pro Leu PheCys Ser Cys Ser His Val Thr Val Val Asp Arg 405 410 415 Ala Val Thr IleThr Asp Leu Arg 420 8 58 PRT Homo sapiens 8 Val Gly Arg Leu Glu Asn AlaIle Gly Trp Tyr His Ser His Pro Gly 1 5 10 15 Tyr Gly Cys Trp Leu SerGly Ile Asp Val Ser Thr Gln Met Leu Asn 20 25 30 Gln Gln Phe Gln Glu ProPhe Val Ala Val Val Ile Asp Pro Thr Arg 35 40 45 Thr Ile Ser Ala Gly LysVal Asn Leu Gly 50 55 9 58 PRT Drosophila melanogaster 9 Val Gly Arg MetGlu His Ala Val Gly Trp Tyr His Ser His Pro Gly 1 5 10 15 Tyr Gly CysTrp Leu Ser Gly Ile Asn Val Ser Thr Gln Met Leu Asn 20 25 30 Gln Thr TyrGln Glu Pro Phe Val Ala Ile Val Val Asp Pro Val Arg 35 40 45 Thr Val SerAla Gly Lys Val Cys Leu Gly 50 55 10 58 PRT Arabidopsis thaliana 10 AlaGly Arg Leu Glu Asn Val Val Gly Trp Tyr His Ser His Pro Gly 1 5 10 15Tyr Gly Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln Arg Leu Asn 20 25 30Gln Gln His Gln Glu Pro Phe Leu Ala Val Val Ile Asp Pro Thr Arg 35 40 45Thr Val Ser Ala Gly Lys Val Glu Ile Gly 50 55 11 58 PRT Caenorhabditiselegans 11 Glu Gly Arg Lys Glu Lys Val Val Gly Trp Tyr His Ser His ProGly 1 5 10 15 Tyr Gly Cys Trp Leu Ser Gly Ile Asp Val Ser Thr Gln ThrLeu Asn 20 25 30 Gln Lys Phe Gln Glu Pro Trp Val Ala Ile Val Ile Asp ProLeu Arg 35 40 45 Thr Met Ser Ala Gly Lys Val Asp Ile Gly 50 55 12 58 PRTArchaeoglobus fulgidus 12 Leu Pro Ile Gly Met Lys Val Phe Gly Thr ValHis Ser His Pro Ser 1 5 10 15 Pro Ser Cys Arg Pro Ser Glu Glu Asp LeuSer Leu Phe Thr Arg Phe 20 25 30 Gly Lys Tyr His Ile Ile Val Cys Tyr ProTyr Asp Glu Asn Ser Trp 35 40 45 Lys Cys Tyr Asn Arg Lys Gly Glu Glu Val50 55 13 58 PRT Pyrococcus horikoshii 13 Met Pro His Asp Glu Ser Ile LysGly Thr Phe His Ser His Pro Ser 1 5 10 15 Pro Phe Pro Tyr Pro Ser GluGly Asp Leu Met Phe Phe Ser Lys Phe 20 25 30 Gly Gly Ile His Ile Ile AlaAla Phe Pro Tyr Asp Glu Asp Ser Val 35 40 45 Lys Ala Phe Asp Ser Glu GlyArg Glu Val 50 55 14 58 PRT Thermoplasma volcanium 14 Lys Pro Ile AspPhe Ser Leu Val Gly Ser Val His Ser His Pro Ser 1 5 10 15 Gly Ile ThrLys Pro Ser Asp Glu Asp Leu Arg Met Phe Ser Leu Thr 20 25 30 Gly Lys IleHis Ile Ile Val Gly Tyr Pro Tyr Asn Leu Lys Asp Tyr 35 40 45 Ser Ala TyrAsp Arg Ser Gly Asn Lys Val 50 55 15 58 PRT Methanobacteriumthermoautotrophicum 15 Leu Pro Pro Phe Thr Gly Ala Val Gly Ser Val HisSer His Pro Gly 1 5 10 15 Pro Val Asn Leu Pro Ser Ala Ala Asp Leu HisPhe Phe Ser Lys Asn 20 25 30 Gly Leu Phe His Leu Ile Ile Ala His Pro TyrThr Met Glu Thr Val 35 40 45 Ala Ala Tyr Thr Arg Asn Gly Asp Pro Val 5055 16 58 PRT Aquifex aeolicus 16 Ile Ser Lys Gly Met Glu Ile Val Gly ValTyr His Ser His Pro Asp 1 5 10 15 His Pro Asp Arg Pro Ser Gln Phe AspLeu Gln Arg Ala Phe Pro Asp 20 25 30 Leu Ser Tyr Ile Ile Phe Ser Val GlnLys Gly Lys Val Ala Ser Tyr 35 40 45 Arg Ser Trp Glu Leu Lys Gly Asp LysPhe 50 55 17 60 PRT Mycobacterium tuberculosis 17 Glu Asp Ala Asp GluVal Pro Val Val Ile Tyr His Ser His Thr Ala 1 5 10 15 Thr Glu Ala TyrPro Ser Arg Thr Asp Val Lys Leu Ala Thr Glu Pro 20 25 30 Asp Ala His TyrVal Leu Val Ser Thr Arg Asp Pro His Arg His Glu 35 40 45 Leu Arg Ser TyrArg Ile Val Asp Gly Ala Val Thr 50 55 60 18 58 PRT Escherichia coli 18Ile Lys Ile Asn Ala Ser Ala Leu Ile Leu Ala His Asn His Pro Ser 1 5 1015 Gly Cys Ala Glu Pro Ser Lys Ala Asp Lys Leu Ile Thr Glu Arg Ile 20 2530 Ile Lys Ser Cys Gln Phe Met Asp Leu Arg Val Leu Asp His Ile Val 35 4045 Ile Gly Arg Gly Glu Tyr Val Ser Phe Ala 50 55 19 57 PRT Drosophilamelanogaster 19 Thr Gly Arg Pro Glu Met Val Val Gly Trp Tyr His Ser HisPro Gly 1 5 10 15 Phe Gly Cys Trp Leu Ser Gly Val Asp Ile Asn Thr GlnGln Ser Phe 20 25 30 Glu Ala Leu Ser Glu Arg Ala Val Ala Val Val Val AspPro Ile Gln 35 40 45 Ser Val Lys Gly Lys Val Val Ile Asp 50 55 20 57 PRTHomo sapiens 20 Thr Gly Arg Pro Glu Met Val Val Gly Trp Tyr His Ser HisPro Gly 1 5 10 15 Phe Gly Cys Trp Leu Ser Gly Val Asp Ile Asn Thr GlnGln Ser Phe 20 25 30 Glu Ala Leu Ser Glu Arg Ala Val Ala Val Val Val AspPro Ile Gln 35 40 45 Ser Val Lys Gly Lys Val Val Ile Asp 50 55 21 57 PRTDictyostelium discoideum 21 Thr Gly Arg Asp Glu Ile Val Ile Gly Trp TyrHis Ser His Pro Gly 1 5 10 15 Phe Gly Cys Trp Leu Ser Ser Val Asp ValAsn Thr Gln Gln Ser Phe 20 25 30 Glu Gln Leu Gln Ser Arg Ala Val Ala ValVal Val Asp Pro Leu Gln 35 40 45 Ser Val Arg Gly Lys Val Val Ile Asp 5055 22 57 PRT Saccharomyces cerevisiae 22 Thr Gly Arg Asp Gln Met Val ValGly Trp Tyr His Ser His Pro Gly 1 5 10 15 Phe Gly Cys Trp Leu Ser SerVal Asp Val Asn Thr Gln Lys Ser Phe 20 25 30 Glu Gln Leu Asn Ser Arg AlaVal Ala Val Val Val Asp Pro Ile Gln 35 40 45 Ser Val Lys Gly Lys Val ValIle Asp 50 55

What is claimed is:
 1. A method of deconjugating a modifier protein froma target protein, wherein the modifier protein is conjugated to thetarget protein via a peptide bond between the carboxy terminus of themodifier protein and a free amino group of the target protein, themethod comprising contacting the target protein to a polypeptidecomprising a subunit characterized as JAB subunit.
 2. The method ofclaim 1, wherein the target protein is a cullin protein.
 3. The methodof claim 2, wherein the target protein is Cul1, Cul2, Cul3, Cul4A,Cul4B, or Cul5.
 4. The method of claim 1, wherein the target protein hasubiquitin ligase activity.
 5. The method of claim 1, wherein the targetprotein is part of a protein complex having ubiquitin ligase activity.6. The method of claim 1, wherein the modifier protein is NEDD8, UBL1,SMT3H2, SMT3H1, APG12, FAT10, Fau, UCRP, URM1, or UBL5.
 7. The method ofclaim 1, wherein the polypeptide is a polypeptide complex ofCOP9/signalsome.
 8. The method of claim 1, wherein the polypeptide isAMSH, AMSH1, or AMSH2.
 9. The method of claim 1, wherein the targetprotein is exposed to the polypeptide in vitro.
 10. The method of claim1, wherein the target protein is exposed to the polypeptide in vivo. 11.A method for screening for an agent that affects deconjugation of amodifier protein from a target protein, wherein the modifier protein isconjugated to the target protein via a peptide bond between the carboxyterminus of the modifier protein and a free amino group of the targetprotein, the method comprising incubating in the presence and absence ofa test agent, the target protein and a polypeptide comprising a subunitcharacterized as JAB subunit, determining the effect of the test agent,wherein an increase or decrease in the amount of the target protein notconjugated to the modifier protein caused by the test agent isindicative of an agent affecting deconjugation of the modifier proteinfrom the target protein.
 12. The method of claim 11, wherein the targetprotein is a cullin protein.
 13. The method of claim 12, wherein thetarget protein is Cul1, Cul2, Cul3, Cul4A, Cul4B, or Cul5.
 14. Themethod of claim 11, wherein the target protein has ubiquitin ligaseactivity.
 15. The method of claim 11, wherein the target protein is partof a protein complex having ubiquitin ligase activity.
 16. The method ofclaim 11, wherein the modifier protein is NEDD8, UBL1, SMT3H2, SMT3H1,APG12, FAT10, Fau, UCRP, URM1, or UBL5.
 17. The method of claim 11,wherein the polypeptide is a polypeptide complex of COP9/signalsome. 18.The method of claim 11, wherein the polypeptide is AMSH, AMSH1, orAMSH2.
 19. The method of claim 11, wherein a test agent decreasing theamount of the target protein not conjugated to the modifier protein isindicative of an agent decreasing deconjugation of the modifier proteinfrom the target protein.
 20. The method of claim 11, wherein the targetprotein has the activity of peroxidase, alkaline phosphatase, orluciferase.
 21. The method of claim 11, wherein the target protein is afluorescent protein.
 22. The method of claim 21, wherein the fluorescentprotein is green fluorescent protein, yellow fluorescent protein, cyanfluorescent protein, or dsRed.
 23. The method of claim 21, wherein thetarget protein is a fluorescent protein via chemical modification. 24.The method of claim 11, wherein the target protein causes production ofa detectable signal upon deconjugation from the modifier protein. 25.The method of claim 11, wherein the polypeptide is a polypeptide complexof 26S proteasome.
 26. The method of claim 11, wherein the polypeptideis a polypeptide complex of 26S proteasome and the modifier protein isan ubiquitin.
 27. The method of claim 25, wherein the incubation isconducted in the presence and absence of the test agent, the targetprotein, the 26S proteasome, and a 20S inhibitor.
 28. The method ofclaim 25, wherein the incubation is conducted in the presence andabsence of the test agent, the target protein, the 26S proteasome, a 20Sinhibitor, and ATP.
 29. The method of claim 27, wherein the incubationfurther includes an inhibitor of deubiquitination by an ubiquitinisopeptidase.
 30. The method of claim 25, wherein the target protein notconjugated to the modifier protein is not degraded.
 31. The method ofclaim 25, wherein the target protein is Sic1.
 32. The method of claim25, wherein the 26S proteasome is purified from S. cerevisiae.
 33. Themethod of claim 25, wherein the 26S proteasome is purified fromeukaryotic cells.
 34. The method of claim 25, wherein the 26S proteasomeis purified from human cells.
 35. An agent identified by the method ofclaim
 11. 36. An agent identified by the method of claim
 19. 37. Anagent identified by the method of claim
 25. 38. A method of increasingconjugation of a modifier protein to a target protein, wherein themodifier protein is conjugated to the target protein via a peptide bondbetween the carboxy terminus of the modifier protein and a free aminogroup of the target protein in a cell, the method comprising inhibitinga polypeptide comprising a subunit characterized as JAB subunit in thecell.
 39. The method of claim 38, wherein the polypeptide isCOP9/signalsome.
 40. The method of claim 38, wherein the polypeptide isAMSH, AMSH1, or AMSH2.
 41. The method of claim 38, wherein thepolypeptide is 26S proteasome.
 42. The method of claim 38, wherein thetarget protein is a cullin protein.
 43. The method of claim 42, whereinthe target protein is Cul1, Cul2, Cul3, Cul4A, Cul4B, or Cul5.
 44. Themethod of claim 38, wherein the target protein has ubiquitin ligaseactivity.
 45. The method of claim 38, wherein the target protein is partof a protein complex having ubiquitin ligase activity.
 46. The method ofclaim 38, wherein the modifier protein is NEDD8, UBL1, SMT3H2, SMT3H1,APG12, FAT10, Fau, UCRP, URM1, or UBL5.
 47. A method of treating acondition selected from the group consisting of neoplastic growth,angiogenesis, infection, chronic inflammation, asthma, ischemia andreperfusion, multiple sclerosis, rheumatoid arthritis, and psoriasiscomprising administering an agent identified by the method of claim 19to a subject in need of such treatment.